Chir99021

CHIR99021 and rabbit anti-β-catenin antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). BIO was purchased from Sigma-Aldrich. β-Actin mouse monoclonal antibody was purchased from Beijing TransGen Biotech Co., Ltd (Beijing, China). Drosha antibody was purchased from Cell signaling (Danvers, MA). Alexa Fluor 555-labeled goat anti-rabbit IgG and anti-rabbit/mouse horseradish peroxidase-conjugated secondary antibody were obtained from the Beyotime Institute of Biotechnology (Jiangsu, China).

To induce IM cells, hiPSC/ESC colonies grown on feeder layers of mitomycin C-treated mouse SNL feeder cells [37] (link) were first dissociated by an enzymatic method with CTK dissociation solution, and incubated on gelatin-coated plates for 30 min to remove SNLs. Then, the cells were dissociated into single cells by gentle pipetting after the treatment with Accutase (Innovative Cell Technologies, Inc.) for 20 min. The cells were then seeded on Matrigel (Matrigel Matrix Growth Factor Reduced, BD)-coated plates at a density of 1.5×10⁵ cells/cm².
For the original small molecule methods, the dissociated hiPSCs/ESCs were treated with 3 µM CHIR99021 and 1 µM AM580 (Santa Cruz Biotechnology) or 1 µM TTNPB (Santa Cruz Biotechnology) in Stage 1 medium for two days. Next, the culture medium was replaced with Stage 2 medium containing 1 µM AM580 or 1 µM TTNPB. The Stage 2 cultures were maintained for an additional three to 12 days. The growth factor method was previously described as a “single-cell method” [19] (link).
For the serum-free small molecule methods, the cells were cultured with a serum-free medium containing DMEM/F12 + Glutamax supplemented with 1×B27 supplement (Invitrogen) and 500 U/ml penicillin/streptomycin on Synthemax (Synthemax II-SC Substrate, Corning)-coated plates throughout the differentiation culture.

CHIR99021 (Santa Cruz, CA, USA) was dissolved in dimethyl sulfoxide (DMSO) and added to cell medium at a final concentration of 3 μM for 24 h unless otherwise specified in J1 mESCs. While F9 ECs were treated with CHIR at a final concentration of 3 μM, 5 μM, 10 μM, 15 μM and 20 μM respectively. Cell medium with an equal volume of DMSO was used as a control.